Netrin-1 Promotes Osteoclast Activity in Multiple Myeloma Under Hypoxic Conditions: Modulation By a RANKL Inhibitor

Myeloma-related bone disease leads to progressive destruction of the skeleton and is the most severe cause of morbidity in multiple myeloma (MM). Although the mechanisms ofosteolytic activity in MM are not fully understood, current evidence points to osteoclast (OC) hyperactivity coupled with bone resorption. Osteoclasts are generated in the bone marrow (BM) by myeloid progenitors through increased levels of NF-κB ligand and M-CSF whose intracellular pathways propagate signals that could activate key transcription factors, resulting in the degradation of the bone matrix. In this context, recent reports on the expression of Netrin-1 in osteoclasts prompted us to further investigate whether secreted Netrin-1 from MM cells contributes to bone-related osteolytic activities in MM patients.  Netrin-1 is a member of the axonal guidance protein family that regulates cell migration and promotes inflammation which subsequently increases macrophage activity in the BM. In contrast to other organs, the BM has long been accepted to be a naturally hypoxic organ. Given that Netrin-1 also is an indicator of oxidative stress, we investigated its potential role in promoting osteoclast activity in MM under hypoxic conditions.

Sample description and assays:  We used (a) U266 MM cells, (b) cells isolated from BM aspirates (mesenchymal stem cells-MSCs) of MM patients and (c) mouse MM cells (5TGM1). Patient samples were obtained with written informed consent and approved by the Institutional Review Board of NYU School of Medicine, and samples from healthy volunteers were used for relevant controls. Cells were exposed to hypoxic conditions (1% oxygen) for 7 days vs. normal oxygen. Reactive oxygen species (ROS) activity assay was measured in cell culture samples in triplicate (100 μl each). To measure Netrin-1 activity, ELISA was performed using a specific antibody. Osteoclast activity and Netrin-1 expression was assessed in cells exposed to hypoxic conditions in the presence of M-CSF and RANKL for 14 days.  After confirming the presence of differentiated osteoclast (>80%) confirmed by TRAPC assays, cells were further treated with the RANKL inhibitor denosumab (30 ng).

Results: Human MM cells refractory to velcade treatment grown under hypoxic conditions for 7 days exhibited a significant increase in proliferation (> 3 fold) compared with the control (0.2/0.56± 0.1-OD) and an increase in migratory colonies in a hyaluronic acid (HA)-based 3D matrix (17/87 > 5 fold increase) in contrast to that observed in a 2D culture showing (5/15) >2 fold. We observed an elevated level of RANKL (350±9 pgs >5 fold compared with that in the control (47±2 pgs). In addition, increased secreted Netrin-1was observed in the cell culture medium. Netrin-1 activity measured by ELISA was enhanced by treatment with TNFα and inhibited by > 5 fold upon treatment with denosumab. Preliminary findings on Crisper-cas-9-directed knockdown of Netrin-1 in M-CSF-RANKL treated pre-osteoclasts will potentially confirm its specific role in promoting osteolytic activities particularly under hypoxia-mediated oxidative stress conditions. The above preliminary findings on decreased pre-osteoclasts associated with a decline in ROS activity and Netrin-1 levels after treatment with denosumab is significant.

Conclusion: Our data provide preliminary evidence on hypoxia-dependent release of Netrin-1 in the promotion of osteoclast activity that is potentially modulated by a RANKL inhibitor in MM refractory patients who are at high risk for bone-related MM.